High molecular weight kininogen (HMWK) is one of four plasma proteins which are involved in the contact system of plasma proteolysis and are modulatory proteins for coagulation, fibronolysis, complement activation, prorenin activation, and possibly other biochemical pathways occurring in the plasma. The other contact proteins are prekallikrein (also known as Fletcher factor), factor XII (also known as Hageman factor) and factor XI (also known as plasma thromboplastin antecedent).
The plasma kininogens are single-chain glycoproteins which are present in human blood plasma and tissues in two forms: high molecular weight kininogen (120 kDa) and low molecular weight kininogen (64 kDa). Both kininogens are parent molecules for the nonapeptide bradykinin, the most potent naturally-occurring vasodilatory substance. High molecular weight kininogen also functions as a cofactor for the activation of the following plasma zymogens: prekallikrein, factor XI, and indirectly, factor XII.
Factor XI in plasma is activated in vitro by surface-bound activated factor XII (factor XIIa). Factor XIIa arises from the interaction of factor XII zymogen with negatively-charged surfaces. The process is amplified by kallikrein-mediated proteolysis of factor XII, and is further amplified when kallikrein is surface-bound via high molecular weight kininogen.
High molecular weight kininogen forms a complex with factor XI and is responsible for transporting the factor XI zymogen to a negatively-charged surface, upon which it is converted to the active enzyme factor XIa by factor XIIa.
The surface-mediated activation of the proteins of the contact phase of plasma proteolysis occurs in certain pathological conditions such as gram negative sepsis, typhoid fever, and acute attacks of hereditary angioedema. Contact activation may also occur during exposure of plasma to foreign surfaces, such as during hemodialysis, extracorporeal circulation, and implantation of artificial organs and grafts.
Present methods for measuring high molecular weight kininogen include (1) functional coagulant assays, (2) immunologic assays for HMWK, and (3) bioassays and immunoassays for the peptide bradykinin. Each method has limitations and/or drawbacks.
The coagulant assay relies on plasma from an individual with a congenital deficiency of high molecular weight kininogen. Less than ten individuals with this defect have been described in the literature, to date:
Fitzgerald - Saito et al, J. Clin. Invest. 55: 1082-1089 PA1 Williams - Colman et al, J. Clin. Invest. 56: 1650-1662 PA1 Flaujeac - Wuepper et al, J. Clin. Invest. 56: 1663-1672 PA1 Washington - Donaldson et al, J. Lab Clin. Med. 87: 327-337 PA1 Reid - Lutcher, Clin. Res. 24: 440A (1976) (abstract) PA1 Fujiwara - Oh-Ishi et al, Adv. Exp. Med. Biol. 120B: 93-99
Coagulation assays, in general, have a high coefficient of variation, and the HMWK coagulant assay relies on rare, congenitally-deficient plasma as substrate.
Immunological determinations for measuring high molecular weight kininogen include rocket electrophoresis: Bouma et al, J. Lab. Clin. Med. 96: 693-709 (1980); radioimmunoassay: Proud et al, J. Lab. Clin Med. 95: 563-574 (1980); Syvanen et al, Febs. Letters 129: 241-245 (1981); Uchida et al, Throm. Res. 15: 127-134 (1979); Kerbiriou-Nabias et al, Brit. J. Haematol. 56: 273-286 (1984); and hemagglutination inhibition: Klenieweski et al, Proc. Soc. Exp. Biol. Med. 156: 113-117 (1977). Such immunological assays for high molecular weight kininogen are time-consuming. They measure the amount of HMWK protein only, without regard to its activity. These methods yield no information concerning the functional integrity of the protein.
While the above-cited hemagglutination inhibition reaction requires less time than other immunoassays, it is laborious and requires many controls. In addition, care must be taken when preparing an antibody to HMWK to assure that it does not cross-react with low molecular weight kininogen, which has the same heavy chain as HMWK.
Bioassays for bradykinin require the sacrificing of an animal and are technically difficult to perform. Radioimmunoassays for bradykinin require special antisera. Damkjaer et al, Clin. Chem. Acta 125: 145-156 (1982); van Rosevelt et al, Clin. Chem. Acta 126: 81-89 (1982); van Leeuwen et al, Clin. Chem. Acta. 127: 343-351 (1983). Most importantly, the available assays for bradykinin cannot distinguish between the high and low molecular weight forms of kininogen. They thus tell nothing of the contact-cofactor function of high molecular weight kininogen.
What is needed is an inexpensive, rapid and reliable assay for determining the level of functional high molecular weight kininogen in human plasma which can be performed in a clinical setting. An assay employing a synthetic substrate has the advantages of being stable as well as reproducible from one laboratory to another. Synthetic chromogenic substrates have been developed to assay a wide variety of enzymes and cofactors. However, since most chromogenic substrates are hydrolyzed by a wide range of proteases, any assay procedure relying on such substrates must be specific for the protein in question.
While a specific chromogenic assay for factor XI in plasma has been reported utilizing a synthetic chromogenic factor XIa substrate, Scott et al, Blood 63: 42-50 (1984), the assay provides no information concerning the level of functional HMWK. Although this assay will give a negative result in the total absence of HMWK, a negative result is not necessarily indicative of the absence of functional HMWK, since the assay will also fail in the absence of factor XII.
Hereinafter, "HMWK" shall mean high molecular weight human kininogen.